Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

All procedures involving animals were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Committee on Use of Live Animals for Teaching and Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong. Human bone marrow samples were obtained from the iliac crest of healthy donors after obtaining informed consent. Protocols were approved by the Institutional Review Board, The University of Hong Kong.

1. Preparation of Rat MSC Cultures

1. Harvest of MSCs from the femur
   1. Autoclave all dissection tools (i.e., fine dissecting scissors, blunt-tipped cutting scissors, and toothed forceps) at 180 °C for at least 2 h prior to use.
   2. Prepare MSC growth medium comprising of minimal essential medium – alpha modification (αMEM) supplemented with 15% fetal bovine serum (FBS) and penicillin/streptomycin (P/S, 1% v/v).
   3. Sacrifice young male Sprague Dawley rats (200-250 g bodyweight) by pentobarbitone overdose (240 mg/kg bodyweight, intraperitoneal).
      NOTE: Marrow samples from different rats should be processed separately.
   4. Place the sacrificed animals in supine position. Clean their abdomen and lower limbs thoroughly with 70% ethanol.
   5. Remove the skin and subcutaneous tissue over the medial thighs using fine dissecting scissors and forceps. Remove the thigh muscles circumferentially until the femur is exposed. Continue this proximally and distally until the knee and hip joints are seen. Disarticulate the femur through the hip and knee joint using blunt-tipped cutting scissors.
      NOTE: Do not transect the femur to expose the marrow cavity at this stage. Transfer intact femurs to a laminar flow tissue culture hood for further processing.
   6. Use blunt tipped cutting scissors to transect the distal and proximal ends of the femur through the metaphysis.
   7. Place a 70 µm cell strainer over a 50 mL conical tube. Insert a 21 G, 10 mL syringe containing phosphate-buffered saline (PBS, 10 mM Na₂HPO₄, pH 7.4) into the exposed femoral canal and flush the marrow content into the conical tube by repeated flushing.
      NOTE: Approximately 20 mL of PBS is used to flush each femur. If the color of the flushed content remains blood-stained and turbid, a larger volume can be used.
   8. Collect the cells by centrifugation at 480 x g for 5 min. Discard the supernatant. Resuspend the cell pellet in 10 mL of MSC growth medium. Plate the cells onto a 10 cm tissue culture dish.Place the tissue culture dishes into a cell incubator (37 °C, 5% CO₂). Record the initial day of plating as day 0.
      NOTE: In all steps involving centrifugation, set the brake for maximal deceleration.
2. Establishment and expansion of MSC colonies
   NOTE: This protocol relies on tissue culture plastic adherence as a means to select for MSCs from within the marrow cavity¹¹. The bone marrow content is allowed to adhere to the tissue culture plastic for 2 days.
   1. On day 2, rinse the culture plates three times with 10 mL of PBS to remove non-adherent cells. Replace the PBS with 10 mL of MSC growth medium after rinsing. Wash the cells with PBS and replenish with MSC growth medium every 3 days.
      NOTE: MSC colonies should be visible by day 6-7 (Figure 2A).
   2. Passage the cells by day 10 by removing the growth medium and rinsing the cells with PBS. Add 1.5 mL of recombinant enzymatic cell dissociation reagent and incubate at 37 °C for 5 min. Add 3 mL of MSC growth medium to neutralize the reaction. Collect the detached cells by centrifugation at 250 x g for 5 min.
   3. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS.
   4. Seed passaged cells at a density of 40,000 cells/cm² into a 10-cm culture plate in MSC growth medium.
      NOTE: Rat MSCs should reach 80-90% confluence within 2 days of passaging (Figure 2B). Cells can be passaged as described in step 1.2.2 for up to 8 passages. MSCs can be characterized by means of immunocytochemistry and their capacity for trilineage differentiation (Figure 3)¹². Only MSC cultures between passages 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment. MSCs of greater passage number adopt a flattened morphology (Figure 2C) and do not produce sufficient numbers of neural progenitors. These cultures should be discarded.

2. Preparation of Human BMSC Cultures

1. Dilute 1 mL of human bone marrow aspirate with 9 mL of MSC growth medium and plate the cells on a 10-cm tissue culture dish. Maintain the cultures in a cell incubator (37 °C, 5% CO₂).
2. Remove the medium after 2 days and gently rinse the cultures three times with 10 mL of PBS to remove non-adherent cells. After the final rinse, remove the PBS and replace it with 10 mL of MSC growth medium. Replenish the growth medium after every 3 days of culture after the PBS rinse.
   NOTE: MSC colonies should be visible by day 6-7. The number of colonies may vary between subjects.
3. Passage the cells on day 10, as described in step 1.2.2. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS. Seed the passaged cells at a density of 40,000 cells/cm² onto a 10 cm culture plate in MSC growth medium.
   NOTE: Human MSCs (Figure 2D) demonstrate a similar morphology to rat MSCs and likewise should reach 80-90% confluence within 2 days of passaging. They should be characterized by means of immunocytochemistry and their capacity for trilineage differentiation¹². As with rat MSCs, human MSCs that are between passage 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment.

3. Hypoxic Preconditioning

1. Disassemble the hypoxia chamber components (i.e., base, lid, trays) after releasing the ring clamp and wipe the individual parts clean with 70% ethanol. Place the chamber components within a laminar flow tissue culture hood for sterilization under UV light for 15 min.
2. Prior to hypoxic preconditioning, remove the medium and rinse the rat and human MSC cultures (sections 1 and 2) with 10 mL of PBS. Replace the PBS with 10 mL of MSC growth medium supplemented with 25 mM HEPES.
   NOTE: The MSCs cultured upon 10 cm dishes should have reached 80-90% confluency prior to be being subject to hypoxic preconditioning.
3. Place the culture dishes within the hypoxia chamber. Reassemble the chamber components and tighten the ring clamp. Flush a gas mixture of 99% N₂/1% O₂ into the chamber at a flow rate of 10 L/min for 5 min.
4. Seal the connecting ends of the hypoxia chamber to ensure there is no gas leakage. Place the chamber inside the cell incubator (37 °C, 5% CO₂) for 16 h.
5. Upon completion of the hypoxic preconditioning, remove the cultures from the chamber in preparation for the subsequent neural progenitor enrichment culture.

4. Neural Progenitor Enrichment Culture

1. Prepare neural progenitor medium comprised of Dulbecco's modified eagle medium/Ham's nutrient mixture F12 (DMEM/F12) supplemented with B27 (2% v/v), basic fibroblast growth factor (bFGF, 20 ng/mL), epidermal growth factor (EGF, 20 ng/mL), and P/S (1% v/v).
2. Detach the hypoxic preconditioned rat/human MSCs, as described in step 1.2.2. Collect the detached cells by centrifugation at 250 x g for 5 min. Quantify the cells within the pellet using a hemocytometer after appropriate dilution in PBS.
3. Resuspend the cells in neural progenitor medium and seed on low-attachment, 6-well plates at a density of 6,000 cells/cm². Place the culture in a cell incubator (37 °C, 5% CO₂) for 12 days. Replenish 75% of the neural progenitor medium every 3 days.
   NOTE: Sizeable non-adherent cell clusters should be observed by day 6-7. By day 10-12, neurospheres with a diameter ≥ 100 µm can be observed (Figure 4). Hypoxic preconditioned MSCs should yield more neurospheres as compared to MSCs cultured under normoxic conditions⁹.
4. Collect neurospheres on day 12 by aspirating them into a 10-mL pipette and transferring them to a 15 mL conical tube. Centrifuge the neurospheres at 250 x g for 5 min.
   NOTE: Neurospheres can be characterized on day 12 for neural progenitor markers, such as nestin and GFAP⁷.

5. Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons

1. Preparation of purified rat DRG neurons
   1. Autoclave all dissection tools (i.e., dissection scissors, forceps, two microdissection forceps, and microdissecting scissors) at 180 °C for at least 2 h prior to use.
   2. Coat 6-well tissue culture plates with poly-D-lysine (PDL, 10 µg/mL in PBS) at 4 °C overnight. Remove the PDL and rinse with 1.5 mL of PBS per well.
   3. Proceed with coating the plates with laminin (10 µg/mL in PBS) at 37 °C for 2 h. Rinse the plates with 1.5 mL of PBS per well.
   4. Prepare DRG neuron maintenance medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1% v/v), nerve growth factor (NGF, 20 ng/mL), and P/S (1% v/v).
   5. Prepare DRG neuron purification medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1%), NGF (20 ng/mL), P/S (1%), fluorodeoxyuridine (FDU, 10 µg/mL), and uridine (10 µg/mL).
   6. Sacrifice pregnant rats at gestational day 14-15 by pentobarbital overdose (240 mg/kg bodyweight, intraperitoneal).
   7. Place the sacrificed animals in supine position. Clean their abdomen thoroughly with 70% ethanol.
   8. Cut the lower abdominal wall of the animal longitudinally using fine dissecting scissors and forceps. Identify and remove the uterus using dissection scissors. Cut the uterine wall to expose and extract the embryos. Transfer the embryos to a sterile, 10 cm culture dish filled with PBS. Place the culture dish on ice.
   9. Transfer the embryo intended for dissection to a sterile, 10 cm culture dish filled with PBS (room temperature) and position it beneath a dissection microscope. Have the embryo in prone position.
      NOTE: The whitish spinal cord and the attached DRGs can be seen over the dorsal aspect of the embryo, through its translucent skin.
   10. Insert microdissecting forceps along either side of the spinal cord and use blunt dissection to begin separating the spinal cord from the surrounding soft tissue. Cut the spinal cord free from the animal using microdissecting forceps along the neck opening and tail stub. Perform further blunt dissection over the ventral aspect of the cord to free it from surrounding soft tissue.
   11. Use microdissecting forceps to remove residual soft tissue over the dorsal aspect of the freed spinal cord.
       NOTE: At this stage, only the spinal cord, nerve roots, and attached DRG should remain.
   12. Detach individual DRGs from their connecting nerve roots using microdissecting forceps. Use a pipette pen attached to a 1 mL tip to transfer the DRGs to a 1.5 mL, sterile centrifuge tube containing PBS.
       NOTE: For each 1.5 mL tube, a maximum of 100 DRGs can be accommodated.
   13. Centrifuge the DRGs at 250 x g for 5 min and resuspend them in recombinant enzymatic cell dissociation reagent (200 µL per tube). Incubate (37 °C, 5% CO₂) for 10 min. Centrifuge the DRGs at 250 x g for 5 min, remove the supernatant, and resuspend in DRG neuron maintenance medium. Dissociate the pellet by gentle trituration using a 200 µL pipette tip. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution.
   14. Seed the cells at a density of 5,000 cells/cm² into PDL/laminin-coated 6-well plates in 1.5 mL of DRG neuron maintenance medium per well. After two days of culture, remove the DRG neuron maintenance medium, rinse with PBS, and replace with DRG neuron purification medium.
       NOTE: For each purification cycle, treat the DRG cultures with purification medium for 2 days, followed by 1 day of incubation in maintenance medium. After 3-4 purification cycles, the removal of all endogenous glia is expected⁷. This should take approximately 14 days. Purified cultures test positive for the neuronal marker TUJ1 and are absent for S100β expression (Figure 5).
2. Generation of Schwann cell-like cells
   1. Prepare glial induction medium comprised of αMEM supplemented with β-Heregulin (100 ng/mL), bFGF (10 ng/ml), platelet-derived growth factor (PDGF-AA, 5 ng/mL), FBS (10%), and P/S (1% v/v).
   2. Plate the neurospheres prepared in section 4 in PDL/laminin-coated 6-well plates at a density of 5-10 spheres per cm² in 1.5 mL of glial induction medium per well. Replace glial induction medium every 2 days after rinsing the cells with PBS.
      NOTE: Cells from seeded neurospheres are seen to migrate outwards by day 2. By day 7, migratory cells have a tapered appearance and should demonstrate immunopositivity for the Schwann cell markers p75 neurotrophin receptor (p75) and S100β⁷. These cells are referred to as SCLCs.
3. Coculture of SCLCs with DRG neurons
   1. Prepare coculture medium comprised of DRG neuron maintenance medium (step 5.1.4) and glial induction medium (step 5.2.1) at a 1:1 volume-to-volume ratio.
   2. Prepare Schwann cell maintenance medium comprised of DMEM/F12 supplemented with FBS (5%), β-Heregulin (10 ng/mL), and P/S (1% v/v).
   3. Remove the culture medium from day 7 SCLCs, rinse them with PBS, and incubate them with 0.5 mL/well of recombinant enzymatic cell dissociation reagent at 37 °C for 5 min. Resuspend the SCLCs in coculture medium.
   4. Quantify the cells using a hemocytometer after an appropriate dilution.
   5. Seed the SCLCs onto purified DRG neuron cultures at a density of 1,000 cells/cm². Maintain the cocultures for 14 days, with medium replacement every 2 days.
      NOTE: During coculture, SCLCs acquired the spindle-like morphology that is typical of mature Schwann cells (Figure 6). These cells persist in their phenotype after the withdrawal of growth factors and are able to myelinate axons in vitro and in vivo^7,10. The positivity of Schwann cell markers (i.e., p75 and S100β) should be monitored by immunofluorescence.
   6. Upon completion of coculture, passage fate-committed Schwann cells, as described in step 1.2.2. Use 0.5 mL of dissociation reagent per well. Quantify the cells using a hemocytometer after an appropriate dilution.
   7. Resuspend fate-committed Schwann cells in Schwann cell maintenance medium at a density of 10,000 cells/cm². Seed cells into PDL/laminin-coated 6-well plates for immunofluorescence.
      NOTE: Cocultures inevitably contain MSCs that that have adopted a fibroblast cell fate⁷. These cells are passaged together with fate-committed Schwann cells upon the completion of the coculture. Schwann cells lying on top of this fibroblast substratum can be easily detached and further expanded following rinsing with "cold jets" of PBS (4 °C), as has been described elsewhere¹³. Fate-committed Schwann cells can be expanded in maintenance medium for 1 month.